ESCHERICHIA-COLI DNA HELICASE-I - CHARACTERIZATION OF THE PROTEIN AND OF ITS DNA-BINDING PROPERTIES

Gene traI of the Escherichia coli F sex factor which encodes DNA/helicase I was subcloned in a λ pL‐based plasmid vector and expressed in a background of pL non‐repressing cells. Neither the non‐repressed pL promoter nor the production of a high level of functional helicase I are toxic. Enzyme purified from this source was studied in the electron microscope. The results show that helicase I binds cooperatively to single‐stranded DNA. DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth‐contoured filament with a diameter of 12.5 ± 0.4 nm and an axial periodicity of 7.0 ± 0.2 nm. In unfixed specimens, discrete particles with axes of 12.7 ± 0.5 nm and 7.2 ± 0.5 nm are visible. They are consistent in size with helicase I monomers (Mr 180000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient. Helicase I free of DNA appears as aggregates. For comparison, a truncated traI. lacking coding for the amino‐terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment. The chimeric gene product (named helicase I del29) retains strand‐separating activity although it fails to show cooperative DNA binding behavior. Judged from the length of the helicase‐I‐specific sequence of this polypeptide. tral is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map. The revised location of traI has implications for understanding distal functions of the transfer operon. Copyright © 1990, Wiley Blackwell. All rights reserved